master's thesis
Improvement of methods for enrichment and analysis of N-glycosylation of membrane proteins

Nina Fekonja (2015)
Sveučilište u Zagrebu
Farmaceutsko-biokemijski fakultet
Zavod za biokemiju i molekularnu biologiju
Metadata
TitleUnapređenje metode za obogaćivanje i analizu N-glikozilacije membranskih proteina
AuthorNina Fekonja
Mentor(s)Olga Gornik (thesis advisor)
Abstract
Analiza glikanskog dijela membranskih glikoproteina postala je važan analitički izazov zbog njihove izuzetne važnosti u međustaničnom prepoznavanju te interakcijama pojedinih molekula s receptorima na staničnoj površini. Brojni pokušaji izolacije membranskih glikoproteina naišli su na niz prepreka s obzirom na nedovoljnu ponovljivost metoda ili pak onečišćenje citosolnim glikoproteinima. Stoga je cilj ovog rada bio potvrditi učinkovitost i ponovljivost metode izolacije membranskih proteina s Triton X-114 i pročišćavanja organskim otapalima u svrhu analize njihove N-glikozilacije. Sama analiza N-glikana provedena je HILIC-UPLC kromatografijom. Korištene su stanice iz THP stanične linije, ali je ponovljivost potvrđena i na stanicama ljudskog mozga, leukocitima i lizosomima. U metodama za pročišćavanje izoliranih membranskih proteina korištena su različita organska otapala pomoću kojih je dobiven precipitat proteina. Organska otapala koja su upotrijebljena bila su aceton, kombinacija aceton/metanol i kloroform/metanol, gdje se posljednja kombinacija pokazala najučinkovitijom, iako sve tri metode daju kromatograme jednakog glikanskog profila. Također je pokazano da početna količina stanica utječe na intenzitet kromatografskih vršaka. Radi potvrde prisutnosti glikanskih struktura u dobivenim uzorcima, sakupljeni su pojedini kromatografski vršci koji su sekvencirani razgradnjom kombinacijom egzoglikozidaza koje otkidaju pojedine monosaharide s nereduktivnih krajeva glikana, a pokazuju određeni stupanj specifičnosti prema kidanju pojedinih veza. Na taj način uspješno su dokazane i identificirane pojedine glikanske strukture. Važnost ove studije je u napretku izolacije membranskih proteina čime je omogućen bolji uvid u njihovu N-glikozilaciju koja je, osim u dijagnostici i prognostici bolesti, danas važna i biofarmaceutskoj industriji zbog toga što glikozilacija ima učinak na fizikalno-kemijska svojstva proteina što se odražava na njihovu učinkovitost i sigurnost.
Keywordsglycosylation membrane proteins N-glycans Triton X-114 organic solvent precipitation HILIC-UPLC exoglycosidase digestion structural identification
Parallel title (English)Improvement of methods for enrichment and analysis of N-glycosylation of membrane proteins
Committee MembersOlga Gornik
Sandra Šupraha Goreta
Miranda Sertić
GranterSveučilište u Zagrebu
Farmaceutsko-biokemijski fakultet
Lower level organizational unitsZavod za biokemiju i molekularnu biologiju
PlaceZagreb
StateCroatia
Scientific field, discipline, subdisciplineBIOMEDICINE AND HEALTHCARE
Pharmacy
Pharmacy
Study programme typeuniversity
Study levelintegrated undergraduate and graduate
Study programmePharmacy
Academic title abbreviationmag. pharm.
Genremaster's thesis
Language Croatian
Defense date2015-06-18
Parallel abstract (English)
Analysis of glycan part of membrane glycoproteins has become an important analytical challenge because of its high importance in intercellular recognition and interactions of molecules with receptors on the cell surface. Numerous attempts to isolate membrane glycoproteins have encountered a number of obstacles due to the lack of reproducibility of the method or pollution with cytosolic glycoprotein. Therefore, the aim of this study was to confirm the efficacy and reproducibility of the method of isolation of membrane proteins with Triton X-114 and its purification with organic solvents for the purpose of analyzing the N-glycosylation. The analysis of N-glycans was performed with HILIC-UPLC chromatography. Used cells were from THP cell lines, but the repeatability was confirmed with human brain cells, white blood cells and lysosomes. In the method for purifying the isolated membrane proteins are used various organic solvents with which we got a protein precipitate. Organic solvents which have been used are acetone, combinations of acetone/methanol and chloroform/methanol, where the last combination came as most effective, although all three methods gave chromatograms with the same glycan profile. It was also found that the initial amount of cells affects the intensity of the chromatographic peak. To confirm the presence of glycan structure in the obtained samples, individual chromatographic peaks were collected and sequenced with combination of exoglycosidases that are tearing certain non-reducing ends of the monosaccharides from glycans. Thus were successfully proven and identified some glycan structures. The importance of this study is progress in isolation of membrane proteins thus providing a better insight into their N-glycosylation that is, except for the diagnosis and prognosis of disease, today very important in biopharmaceutical industry because glycosylation has an effect on the physical and chemical properties of the protein which is reflected in their effectiveness and safety.
Parallel keywords (Croatian)glikozilacija membranski proteini N-glikani Triton X-114 precipitacija organskim otapalima HILIC-UPLC digestija egzoglikozidazama strukturna identifikacija
Resource typetext
Access conditionOpen access
Terms of usehttp://rightsstatements.org/vocab/InC/1.0/
URN:NBNhttps://urn.nsk.hr/urn:nbn:hr:163:054979
CommitterPetra Gašparac